Adipose Derived Stem Cell (ADSC) Retrieval procedure:

Extract, adipose

Laser or External Ultrasonic Liposuction

Wash aspirates, with sterile phosphate-buffered saline (PBS)

Treat aspirates with 0.075% collagenase in PBS for 30 min at 37°C with gentle agitation; inactivate collagenase with an equal volume of DMEM/10% fetal bovine serum (FBS) and the infranatant centrifuged for 10 min at low speed

Culture processed lipoaspirate in 72 h before analysis

Isolate based on: CD13, CD44, CD49d, CD106, STRO-1 (?)

Go to Culture procedure for pluripotent cells

Peripheral Blood Stem Cells (PBSC) procedure:

Extract (PBSC)

Retrieve 100 ml/cc whole blood

Centrifuge and remove red blood cells

Isolate stem cells with CD34+ antigens from blood source

Culture and Preservation procedures:

Culture

Culture stem cells to more than 400 times original number

Cryopreservation

Add trehalose cryoprotectant

Documentation

Photograph stem cells with inverted microscope, Hoffman optics

Save a 2D electrophoresis image of a sample

Divide 75% of stem cells into 20 or greater 1 cc vials

Place 75% of white blood cells in 1 cc vials

Cool vials at 1 degree centigrade per minute to –100 C

Place in dry –150 C, or –196 C liquid nitrogen

Lyophilization

Use remainder of cells for nuclear freeze-dry procedure

Backup

Distribute cell samples to multiple facilities

Sequence and document

Sequence DNA including mitochondrial and document on intranet, including donor videos and medical records